Inotropic ADP and ATP analogues and their pharmaceutical compositions

ABSTRACT

Purine-containing compounds that modulate cardiac muscle contractility are provided, as well as methods for modulating cardiac muscle contractility and receptors that bind the compounds.

GOVERNMENT SUPPORT

The inventor has been supported by National Institutes of Health GrantsHL44188 and HL48225.

FIELD OF THE INVENTION

This invention relates to compounds that modulate cardiac musclecontractility, to methods for modulating cardiac muscle contractility,and to receptors that bind the compounds.

BACKGROUND OF THE INVENTION

Positive inotropic agents (i.e., agents which increase the contractilityof cardiac muscle in a dose dependent manner) find use, inter alia, inthe treatment of congestire heart failure and as vasodialators.Representative of the three classes of positive inotropic agents are theNa⁺ /K⁺ ATPase inhibitor digitalis, the β-adrenergic agonists dobutamineand dopamine, and the phosphodiesterase inhibitor amrinone.

Each of these classes of positive inotropic agents suffers fromsignificant limitations. Digitalis displays a weak positive inotropiceffect with narrow a therapeutic index, many adverse side effects, andundesirable interactions with other cardiac drugs. Dobutamine anddopamine cause desensitization of the β-adrenergic receptor-mediatedpositive inotropic response, are arrhythmogenic, and can only beadministered intravenously. Orally active β-adrenergic agonists are onlyeffective for a short period of time and lose efficacy due todesensitization. Phosphodiesterase inhibitors, such as milrinone, arepotentially arrhythmogenic and have increased mortality relative todigitalis.

ATP is known to cause an inotropic effect in the heart, which is thoughtto be mediated by the P2 purinergic receptor (P2PR). To date there hasbeen no detailed characterization of the specific P2PR involved, and nosuitable cell model exists for the characterization of P2PR.

Consequently, there is a need in the art for positive inotropic agentswhich overcome the disadvantages associated with known agents, as wellas a need for further information on the mechanisms and receptorsassociated with cardiac muscle contractility.

OBJECTS OF THE INVENTION

It is one object of the present invention to provide inotropic agents,that is, compounds that modulate (i.e., increase or decrease) cardiacmuscle contractility.

It is another object of the invention to provide positive inotropicagents.

It is a further object to provide positive inotropic agents that have abroader therapeutic index than those currently available.

It is yet another object to provide positive inotropic agents havinglonger and more evenly sustained rates of release than those currentlyavailable.

It is a further object to provide positive inotropic agents havinglonger duration of action than those currently available.

It is yet another object to provide inotropic agents that are orallyactive.

It is a further object to identify and characterize receptors that bindthe inotropic agents of the invention.

SUMMARY OF THE INVENTION

These and other objects are accomplished by the present invention, whichprovides novel inotropic agents. In one aspect, the invention providespurine-containing inotropic agents having formula I or II: ##STR1##wherein: R₁ and R₂, independently, are halogen or --R₆ --(R₇)_(p) --R₈ ;

R₃ is H, halogen or --R₆ --(R₇)_(p) --R₈ ;

R₄ is OH or SH;

R₅ is OH or acetamido;

R₆ is NH or S;

R₇ is alkylene having from 1 to 10 carbon atoms;

R₈ is H, NH₂, CN, cycloalkyl having 3 to about 10 carbon atoms, or arylhaving 3 to about 20 carbon atoms;

X and Y are independently N or CH;

n is 0 or 1; and

p is 0 or 1.

In certain preferred embodiments R₈ is --C₆ H₁₁, --C₅ H₉, --C₆ H₅, --C₆H₄ --NO₂, or --CH C₆ H₄ (CH₃)! C₆ H₃ (OCH₃)₂ !. In other preferredembodiments R₃ is H, and R₁ and R₂ are NH₂, S or Cl, and in furtherpreferred embodiments X and Y are N or CH.

The invention also provides compositions comprising the compound offormula I or II and a pharmaceutically acceptable carrier, adjuvant, orvehicle, preferably in an amount effective to increase cardiac muscletissue contractility.

In another aspect, the invention provides compositions comprising thecompounds of the invention, and methods for increasing contractility incardiac muscle tissue. The methods generally comprise contacting cardiacmuscle tissue with a compound of the invention and, optionally,measuring a rate of contraction associated therewith.

The invention also provides methods for increasing cellular contraction,comprising contacting a mammalian cell with a compound of the inventionand, optionally, measuring a rate of contraction associated with thecell. Also provided are methods for treating a mammal in need ofincreased cardiac muscle contractility, comprising administering to themammal a compound of the invention.

In a further aspect, the invention also provides isolated P2_(y) -likepurinergic receptors and compositions comprising a compound of theinvention bound to P2_(y) -like purinergic receptors.

BRIEF DESCRIPTION OF THE FIGURES

The numerous objects and advantages of the present invention can bebetter understood by those skilled in the art by reference to theaccompanying figures, in which:

FIGS. 1a-1d show the positive inotropic response of cardiac ventricularmyocytes to ATP and P₂ purinergic receptor agonists.

FIGS. 2a and 2b show intact myocyte binding with the P_(2y) -selectiveradioligand ³⁵ S!ADPβS.

FIGS. 3a, 3b, 4a, and 4b show structure-activity relationship studieswherein EC₅₀ values determined for agonists stimulating myocytecontractility are compared to the K_(i) of the same agonists ininhibiting high-affinity ³⁵ S! ADPβS binding.

FIG. 5 shows the preparation of derivatives of 3'- amino-3' deoxy-ATP.

FIGS. 6a and 6b show the synthesis of 2-substituted ATP derivatives.

DETAILED DESCRIPTION OF THE INVENTION

It has been found in accordance with the present invention that a classof purine-containing compounds act as inotropic agents and, moreover, aspositive inotropic agents. In certain embodiments, the compounds haveformula I or II: ##STR2## wherein: R₁ and R₂, independently, are halogenor --R₆ --(R₇)_(p) --R₈ ;

R₃ is H, halogen or --R₆ --(R₇)_(p) --R₈ ;

R₄ is OH or SH;

R₅ is OH or acetamido;

R₆ is NH or S;

R₇ is alkylene having from 1 to 10 carbon atoms;

R₈ is H, NH₂, CN, cycloalkyl having 3 to about 10 carbon atoms, or arylhaving 3 to about 20 carbon atoms;

X and Y are independently N or CH;

n is 0 or 1; and

p is 0 or 1.

Alkyl groups according to the invention include but are not limited tostraight chain and branched chain hydrocarbons such as methyl, ethyl,propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl,dodecyl, isopropyl, 2-butyl, isobutyl, 2-methylbutyl, isopentyl,2-methylpentyl, 3-methylpentyl, 2-ethylhexyl and 2-propylpentyl groupshaving 1 to about 12 carbon atoms, preferably 1 to about 6 carbon atoms.Cycloalkyl groups are cyclic alkyl groups such as cyclopropyl,cyclopentyl and cyclohexyl groups. Alkylene groups are straight chain orbranched chain hydrocarbons that are covalently bound to two othergroups. Preferred alkylene groups have the formula -(CH₂)_(n) -where nis 1 to about 12, preferably 1 to about 6, including methylene (n=1) andethylene (n=2) groups. The alkyl and alkylene groups of the inventioncan substituted with a wide variety of moieties and/or internallyinterrupted with heteroatoms, such as O, N, or S.

Aryl groups according to the invention are aromatic groups having 3 toabout 20 carbon atoms, preferably from 3 to about 10 carbon atoms,including, for example, benzyl, imidazolyl, naphthyl, phenyl, pyridyl,pyrimidinyl, and xylyl groups and substituted derivatives thereof,particularly those substituted with alkyl, alkoxy, amino, and nitrogroups.

The term halogen as used herein is intended to denote substituentsderived from fluorine, chlorine, bromine, or iodine.

Preferred groups having formula --R₆ -(R₇)_(p) -R₈ include thefollowing: --NH₂ , --NH--CH₃, --NH--C₂ H₅, --Cl, --S--CH₃, --NH--CH₂--NH₂, --NH--(CH₂)₂ --NH₂, --NH--(CH₂)₃ --NH₂, --NH--(CH₂)₄ --NH₂,--NH--(CH₂)₅ --NH₂, --NH--(CH₂)₆ --NH₂, --S--(CH₂)₆ --CN, --S--CH₂ --C₆H₄ --NO₂, --S--CH₂ --CN, --S--(CH₂)₂ --CN, --S--(CH₂)₄ --CN, --S--(CH₂)₅--CN, --NH--C₆ H₅, --NH--CH₂ C₆ H₅, --NH--(CH₂)₂ --C₆ H₅, --NH--(CH₂)₃--C₆ H₅, --NH--(CH₂)₄ --C₆ H₅, --NH--(CH₂)₅ --C₆ H₅, --NH--(CH₂)₆ --C₆H₅, --NH--C₆ H₁₁, --NH--C₆ H₁₁, --NH--C₅ H₉, --NH--CH₂ -CH C₆ H₄ (CH₃) !C₆ H₃ (OCH₃)₂ !.

While not wishing to be bound by a particular theory, the compounds ofthe invention are believed to exert their positive inotropic effect bymechanisms different from those of other known positive inotropicagents. This mechanism is believed to involve binding to a novel P2_(y)-like purinergic receptor. As used herein, a P2_(y) -like purinergicreceptor is one which upon activation leads to an increase in calciumentry and myocyte contractility subsequent to the activation of a novelmechanism.

The compounds of the invention contain amino groups and, therefore, arecapable of forming salts with various inorganic and organic acids. Suchsalts are also within the scope of this invention. Representative saltsinclude inorganic addition salts such as phosphate, hydrochloride,hydrobromide, hydroiodide, hemisulfate, sulfate, bisulfate and nitrate,and organic salts including, for example, acetate, benzoate, butyrate,citrate, fumarate, heptanoate, hexanoate, lactate, maleate, succinateand tartrate. The salts can be formed by conventional means, such as byreacting the free base form of the product with one or more equivalentsof the appropriate acid in a solvent or medium in which the salt isinsoluble, or in a solvent such as water which is later removed in vacuoor by freeze drying. The salts also can be formed by exchanging theanions of an existing salt for another anion on a suitable ion exchangeresin.

The present invention also provides prophylactic, diagnostic, andtherapeutic compositions comprising one or more compounds of theinvention. By administering an effective amount of such compositions,for example, prophylactic or therapeutic responses can be produced in ahuman or some other type mammal. It will be appreciated that theproduction of prophylactic or therapeutic responses includes theinitiation or enhancement of desirable responses, as well as thecessation or suppression of undesirable responses.

Compositions of the invention can be administered in unit dosage formand may be prepared by any of the methods well known in thepharmaceutical art, for example, as described in Remington'sPharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980). Thecompositions can be in the form of a solid, semisolid or liquid form andcan include one or more of the compounds of the invention as an activeingredient in admixture with an organic or inorganic carrier orexcipient suitable, for example, for oral administration, parenteraladministration, intranasally or dermally, via, for example, trans-dermalpatches. Other suitable modes of administration will be apparent tothose skilled in the art. The active ingredient can be compounded, forexample, with the usual non-toxic, pharmaceutically acceptable carriersfor tablets, pellets, capsules, suppositories, solutions, emulsions,suspensions, and any other form suitable for use. The carriers which canbe used are water, glucose, lactose, gum acacia, gelatin, mannitol,starch paste, magnesium trisilicate, talc, corn starch, keratin,colloidal silica, potato starch, urea and other carriers suitable foruse in manufacturing preparations, in solid, semisolid, or liquid form,and in addition auxiliary, stabilizing, thickening and coloring agentsand perfumes maybe used. The active ingredient is included in thepharmaceutical composition in an amount sufficient to produce thedesired effect upon the process or condition of diseases.

For oral administration, tablets containing various excipients such asmicrocrystalline cellulose, sodium citrate, calcium carbonate, dicalciumphosphate and glycine may be employed along with various disintegrantssuch as starch and preferably corn, potato or tapioca starch, alginicacid and certain complex silicates, together with granulation binderslike polyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate andtalc are often very useful for tabletting purposes. Solid compositionsof a similar type may also be employed as fillers in gelatin capsules;preferred materials in this connection also include lactose or milksugar as well as high molecular weight polyethylene glycols. Whenaqueous suspensions and/or elixirs are desired for oral administration,the active ingredient may be combined with various sweetening orflavoring agents, coloring matter or dyes, and, if so desired,emulsifying and/or suspending agents as well, together with suchdiluents as water, ethanol, glycerin and various like combinationsthereof.

For parenteral administration, solutions containing the compounds of theinvention in either sesame or peanut oil or in aqueous propylene glycolcan be employed. The aqueous solutions should be suitably buffered(preferably pH>8) if necessary and the liquid diluent first renderedisotonic. These aqueous solutions are suitable for intravenous injectionpurposes. The oily solutions are suitable for intra-articular,intra-muscular and subcutaneous injection purposes. The preparation ofall these solutions under sterile conditions is readily accomplished bystandard pharmaceutical techniques well-known to those skilled in theart. Additionally, it is possible to administer the compounds of thepresent invention topically when treating inflammatory conditions of theskin and this may preferably be done by way of creams, jellies, gels,pastes, ointments and the like, in accordance with standardpharmaceutical practice.

The inotropic agents of the invention can be employed as the sole activeagent in a pharmaceutical or can be used in combination with otheractive ingredients, e.g., other positive inotropic agents useful indiseases or disorders.

The amount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. The specificdose level for any particular patient will depend on a variety offactors including the activity of the specific compound employed, theage, body weight, general health, sex, diet, time of administration,route of administration, rate of excretion, drug combination, and theseverity of the particular disease undergoing therapy. In someinstances, dosage levels below the lower limit of the aforesaid rangemay be more than adequate, while in other cases still larger doses maybe employed without causing any harmful side effects provided that suchhigher dose levels are first divided into several small doses foradministration throughout the day. The concentrations of the compoundsdescribed herein found in therapeutic compositions will vary dependingupon a number of factors, including the dosage of the drug to beadministered, the chemical characteristics (e.g., hydrophobicity) of thecompounds employed, and the route of administration. In general terms,the compounds of this invention may be provided in an aqueousphysiological buffer solution (for example, 1 cc) containing about 0.2%w/v compound for oral administration. Typical dose ranges are from about285 μg/kg of body weight per day in three divided doses; a preferreddose range is from about 42 μg/kg to about 171 μg/kg of body weight perday. The preferred dosage of drug to be administered is likely to dependon such variables as the type and extent of progression of the diseaseor disorder, the overall health status of the particular patient, therelative biological efficacy of the compound selected, and formulationof the compound excipient, and its route of administration, as well asother factors, including bioavailability, which is in turn influenced byseveral factors. For example, if the compound is metabolized in theliver or excreted in bile, some of the active compound absorbed from thegastrointestinal tract will be inactivated by the liver before it canreach the general circulation and be distributed to its sites of action.It is not believed that the compounds of the invention will be subjectto this first-pass loss. Additionally, because the instant compounds arepolar and water soluble, it is expected that they will have a smallvolume of distribution, and thus be readily eliminated by the kidney.Moreover, binding of the instant compounds to plasma proteins may limittheir free concentrations in tissues and at their locus of action sinceit is only the unbound drug which equilibriums across the membranereceptor sites. It is anticipated that the phosphate moiety of theinstant compounds may facilitate binding of the compounds to plasmaalbumins, which will in turn influence the amount of free compoundavailable to activate muscle cell P2 purinergic receptors. However, itis expected that such binding to plasma protein will not generally limitrenal tubular secretion of biotransformation since these processes lowerthe free drug concentration and this is rapidly followed by theassociation of this drug-protein complex. Another factor affectingbioavailability is the distribution of the compounds to tissues. Giventhe relatively small size of the compounds and their water solubility,it is anticipated that the compounds will have a relatively fast secondphase of drug distribution. This distribution is determined by both theblood flow to the particular tissue of the organ, such as the heart, aswell as the rate at which the compounds diffuse into the interstitialcompartment from the general circulation through the highly permeablecapillary endothelial (except in the brain). Due to the relativehydrophilicity of these compounds, it is anticipated that there will beno fat or other significant tissue reservoir of the compounds whichwould account for a third phase of distribution-accumulation.

Calculation of the above dosages are based on the molecular weight ofthe compounds, which range from 500-1000 gram per mole, and upon invitro determinations of K_(d) values for the compounds for the P2purinergic receptors, which range from 30-100 nM. For example, assuminga volume of distribution of 15-20 liters for a 70 kg subject, a Kd of100 nM, and a volume of distribution of 20 liters, a 2 mg loading dosewould be required to achieve the Kd value in the extracellular fluidcompartment. The typical and preferred dose ranges were then calculatedbased on all the variables as well as the factors discussed above.

In certain embodiments, methods according to the invention involvecontacting a compounds having formula I or II with cardiac muscletissue. As used herein, the term "contacting" means directly orindirectly causing placement together of moieties to be contacted, suchthat the moieties come into physical contact with each other. Contactingthus includes physical acts such as placing the moleties together in acontainer, or administering moieties to a patient.

The compounds of the invention also can be used as research reagents.The compounds, for example, can be used as synthetic intermediates inthe preparation of nucleosides, nucleotides, and oligonucleotides. Forexample, the compounds of the invention can be functionalized asphosphoramidites and used in automated oligonucleotide syntheses such asthose associated with the well-known polymerase chain reaction (PCR)procedure.

The invention also includes receptor polypeptides (e.g., P2_(y) -likereceptor polypeptides) from any naturally occurring source, preferablymammalian, more preferably human, which exhibit biological activity. Inthe context of the invention, biological activity includes binding to acompound of the invention or otherwise interacting with such a compoundto facilitate or ultimately produce an inotropic response. Polypeptidesalso include homologous sequences (as defined below); allelicvariations; natural mutants; induced mutants; proteins encoded by DNAwhich hybridizes under high or low stringency conditions to P2_(y) -likereceptor encoding nucleic acids retrieved from naturally occurringmaterial; and polypeptides or proteins retrieved by antisera to theP2_(y) -like receptor, especially by antisera to the active site orbinding domain of the P2_(y) -like receptor. The invention also providesother polypeptides, e.g., fusion proteins, which include P2_(y) -likereceptor polypeptides or biologically active fragments thereof.

P2_(y) -like receptor polypeptides will generally exhibit at least about70%, more preferably about 80%, more preferably 90%, still morepreferably 95%, or even 99%, homology (as defined herein) with all orpart of a naturally occurring P2_(y) -like receptor sequence. For thepurposes of determining homology the length of comparison of sequenceswill generally be at least 8 amino acid residues, usually at least about20 amino acid residues, more usually at least about 24 amino acidresidues, typically at least 28 amino acid residues, and preferably morethan about 35 amino acid residues.

The present invention also provides for analogs of P2_(y) -like receptorpolypeptides. Analogs can differ from naturally occurring P2_(y) -likereceptor polypeptides by amino acid sequence differences or bymodifications which do not affect sequence, or by both.

Modifications (which do not normally alter primary sequence) include invivo or in vitro chemical derivatization of polypeptides, e.g.,acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps. Representative modifications include exposing thepolypeptide to enzymes which affect glycosylation, such as mammalianglycosylating or deglycosylating enzymes. Also embraced are sequenceswhich have phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine.

Included are peptides which have been modified so as to improve theirresistance to proteolytic degradation or to optimize solubilityproperties. Analogs can differ from naturally occurring P2_(y) -likereceptor polypeptides by alterations of their primary amino acidsequence. These peptides include genetic variants, both natural andinduced. Induced mutants can be made by various techniques, e.g., byrandom mutagenesis of the encoding nucleic acids using irradiation orexposure to ethanemethylsulfate (EMS), or by site-specific mutagenesisor other techniques of molecular biology. See, Sambrook, Fritsch andManiatis (1989), Molecular cloning: A Laboratory Manual (2d ed.), CSHPress. Also included are analogs which include residues other thannaturally occurring L-amino acids, e.g., D-amino acids or non-naturallyoccurring or synthetic amino acids such as β- or γ-amino acids. Thepeptides of the invention are not limited to products of any of thespecific exemplary process listed herein.

In addition to substantially full-length polypeptides, the presentinvention provides biologically active fragments of the polypeptides. AP2_(y) -like receptor polypeptide (or fragment) is biologically activeif it exhibits a biological activity of a naturally occurring P2_(y)-like receptor polypeptide. Such biological activities include theability to bind, e.g., specifically bind, a compound of the invention.The affinity of a P2_(y) -like receptor polypeptide fragment for apositive inotropic agent of the present invention preferably is at least1% of (more preferably at least 10% or, yet more preferably at least 50%of, still more preferably at least equal to) the affinity of a naturallyoccurring P2_(y) -like receptor polypeptide for a compound of theinvention). Another biological activity is the ability to bind to anantibody which is directed at an epitope which is present on a naturallyoccurring P2_(y) -like receptor, preferably an epitope on a P2_(y) -likereceptor domain of naturally occurring P2_(y) -like receptor.

Putative biologically active fragments of P2_(y) -like receptors can begenerated by methods known to those skilled in the art. The ability of acandidate fragment to bind a positive inotropic agent of the inventioncan be assessed by methods known to those skilled in the art.

The invention also provides nucleic acid sequences, and purifiedpreparations thereof, which encode the P2_(y) -like receptorpolypeptides described herein.

The invention also provides antibodies, preferably monoclonalantibodies, which bind specifically to P2_(y) -like receptorpolypeptides and preferably antibodies which bind to the agonist bindingdomain of a P2_(y) -like receptor polypeptide.

As used herein, the term "fragment or segment", as applied to apolypeptide, will ordinarily be at least about 5 contiguous amino acids,typically at least about 10 contiguous amino acids, more typically atleast about 20 contiguous amino acids, usually at least about 30contiguous amino acids, preferably at least about 40 contiguous aminoacids, more preferably at least about 50 contiguous amino acids, andeven more preferably at least about 60 to 80 or more contiguous aminoacids in length.

As used herein, the term "substantially pure" describes a compound(e.g., a protein or polypeptide such as a P2_(y) -like receptor proteinor polypeptide) which has been separated from components which naturallyaccompany it. Typically, a compound is substantially pure when at least10%, more preferably at least 20%, more preferably at least 50%, morepreferably at least 60%, more preferably at least 75%, more preferablyat least 90%, and even more preferably at least 99%, of the totalmaterial (by volume, by wet or dry weight, or by mole percent or molefraction) in a sample is the compound of interest. Purity can bemeasured by any appropriate method. In the case of polypeptides, forexample, purity can be measured by column chromatography, polyacrylamidegel electrophoresis, or HPLC analysis. A compound such as a protein isalso substantially purified when it is essentially free of naturallyassociated components or when it is separated from the nativecontaminants which accompany it in its natural state.

A "substantially pure nucleic acid", as used herein, refers to a nucleicacid sequence, segment, or fragment which has been purified from thesequences which flank it in a naturally occurring state, e.g., a DNAfragment which has been removed from the sequences which are normallyadjacent to the fragment such as the sequences adjacent to the fragmentin a genome in which it naturally occurs. The term also applies tonucleic acids which have been substantially purified from othercomponents which naturally accompany the nucleic acid, e.g., RNA or DNA,which has been purified from proteins which naturally accompany it inthe cell.

"Homologous", as used herein, refers to the subunit sequence similaritybetween two polymeric molecules, e.g., between two nucleic acidmolecules such as two DNA molecules, or two polypeptide molecules. Whena subunit position in both of the two molecules is occupied by the samemonomeric subunit (e.g., if a position in each of two DNA molecules isoccupied by adenine) then they are homologous at that position. Thehomology between two sequences is a direct function of the number ofmatching or homologous positions. For example, if 5 of 10 positions intwo compound sequences are matched or homologous then the two sequencesare 50% homologous, if 9 of 10 are matched or homologous, the twosequences share 90% homology. By way of example, the DNA sequences 3'ATTGCC 5' and 3' TTTCCG 5' share 50% homology.

Complex or affinity complex, as used herein, refers to an association ofa first and a second component, e.g., a receptor and its ligand. Theassociation can include either or both covalent and noncovalent bonds.

Thus, the invention provides a novel receptor capable of binding theinotropic agents of the invention.

Additional objects, advantages, and novel features of this inventionwill become apparent to those skilled in the art upon examination of thefollowing examples thereof, which are not intended to be limiting.

EXAMPLE 1

P2 purinergic receptor subtypes, including P2_(y) and P2_(u), have beencloned from chick brain, a mouse-derived cell line and rat brain.Alignment of the sequences from these distinct P2 purinergic receptorsrevealed that the transmembrane III region displays a high degree ofamino acid identity (>70%) that is not seen elsewhere in the sequence.This high degree of homology was well-preserved from chick to mouse orrat. Such data are consistent with the notion that transmembrane III isfunctionally important in the family of P2 purinergic receptors.

Two separate oligonucleotides were made which are identical to twostretches of sequence in the transmembrane III region. Both are used toscreen a cDNA library (λgt10 phage library) constructed from rat andchick heart. A suitable library is available, for example, fromClontech, Palo Alto, Calif. Phages that contain hybridizing cDNA's areisolated and purified and the cDNA insert corresponding to cardiac P2purinergic receptor is sequenced. The sequence is compared to the knownP2 purinergic receptor sequences using a computer-based analysis(GeneWorks, Intelligenetics) to initially classify the cardiac sequence.Confirmation of the identity of the cardiac sequence as a cardiac P2_(y)purinergic receptor is obtained by expressing the sequence using aneukaryotic expression vector in COS cell followed by pharmacologicalcharacterization using known P2 receptor subtype-selective agonists.Expression is carried out in the cultured chick heart cells to discernan enhanced contractile response of the transfected myocyte to P2purinergic agonist using the method of Xu, H., Miller, J. and Liang,B.T., Nucleic Acids Research 20(23) 6425-6426, (1992). Once such a cloneencoding the cardiac-like receptor is obtained, the DNA is subclonedinto the multiple cloning site of the expression vector pBlueBac-His inframe with the ATG on the leader peptide. The recombinant vector isexpressed in the baculovirus/insect cell system to produce a largequantity of the P2 purinergic protein using standard procedures.Suitable procedures include Rotrosen, D., Yeung, C., Katkin, J., J.Biol. Chem. 268:14256-14260 (1993); Gui, J., Lane, W.S. Fu, X. D.,Nature 369:6482 (1994); and Roddy, R., Yoshimoto, T., Yamamoto, S.,Funk, C., Marnett, L.J., Arch. Biochem. Biophys. 312:219-226 (1994).

The receptor protein thus isolated and purified can then be used for avariety of purposes such as antibody generation, mutagenesis,glycosylation, phosphorylation of methylation of the receptor,substitution of a particular amino acid residue of the receptor, andreconstitution in vitro phospholipid system for interaction with thecompound of the invention.

The receptor is additionally characterized with respect to itspharmacological binding properties, density and regulation by, forexample, using a radioligand selective for the cardiac P2_(y) -likereceptor. See for Example 3, infra, and FIGS. 2a and 2b.

EXAMPLE 2

Demonstration of a positive inotropic response of cardiac ventricularmyocytes to ATP and P₂ purinergic receptor agonists.

Cardiac ventricular myocytes were prepared and changes in thecontractile amplitude were determined in response to the agonist(s).

A. Cell Isolation Procedure

The cell isolation procedure is that of Kelly, R.A., Eid, H., Kramer,B.K., O'Neill, M., Liang, B.T., Reefs, M., Smith, T.W., J. Clin. Invest.86:1164-1171 (1990), and Sen, L., Liang, B.T., Colucci, W.S., Smith,T.W., Circ. Res. 67:1182-1192 (1990), modified to include the use of ahigh-speed peristaltic pump (Masterflex from Cole-Palmer/Spectrum),choice of lots of collagenase, hyaluronidase and protease that give thehighest proportion of rod-shaped myocytes, inclusion of bovine serumalbumin containing various enzymes, and preplating with laminin-coatedplates or glass coverslips (for contractility measurement) to eliminatedamaged, rounded up myocytes. Specifically, hearts from Sprague-Dawleyrats are perfused in a retrograde manner for 5 minutes inKrebs-Henseleit (KH) bicarbonate buffer containing 118 mM NaCl, 4.6 mMKCl, 1.2 mM MgSO₄, 1.25 mM CaCl₂, 1.2 mM KH₂ PO₄, 25mM NaHCO₃, and 11 mMglucose, gassed with 95% O₂ /5% CO₂ / at pH 7.4 (37° C., and anosmolality of 287 mosm/liter). The perfusion buffer is changed tonominally Ca²⁺ -free KH buffer for 5 min to arrest spontaneous beating.The hear is then perfused with the Ca² +-free KH buffer containing 0.05%collagenase (Worthington) and 0.03% hyaluronidase (Sigma Chem. Co., St.Louis, Mo.) for 20 min. After removing the aria and the great vessels,the ventricular tissue is finely minced in the samecollagenase/hyaluronidase buffer described above except that trypsin(0.02 mg/ml, Sigma) and deoxyribonuclease (0.02 mg/ml, Sigma) are alsoadded. The minced tissue is further incubated in this buffer at 37° C.with shaking to facilitate dissociation of individual ventricularmyocytes. The dissociated cells containing some damaged cells arefiltered and layered twice over a 6% bovine serum albumin gradient. Therod-shaped healthy heart cells sediment more easily than the rounddamaged cells. After the second sedimentation through the 6% BSAgradient, the final pellet should contain more than 90% rod-shaped heartcells.

B. Cardiac Cell Preparation

Ventricular myocytes were cultured from chick embryos 14 days in ovoaccording to previously describe procedure (7). Briefly, isolatedventricular myocytes from chick embryos 14 in ovo were prepared incalcium- and magnesium-free Hanks' balanced salt sodium (HBSS)containing 0.025% trypsin (GIBCO, Grand island, N.Y.). Afterneutralization of trypsin medium containing horse serum and HBSS, cellswere centrifuged and resuspended in culture medium containing 6% fetalbovine serum, 40% Medium 199 (GIBCO), 0.1% penicillin/streptomycin, anda salt solution. The final concentrations in the culture medium (inmmol/L) were Na 142, K 3.3, Mg 0.7, Ca 1.4, Cl 130, HCO₃ 16.4, andglucose 5.5. Cells were plated at a density of 400,000 cell per ml andcultivated in a humidified 5% CO₂ -95% air mix at 37° C.. Cells becameconfluent on day 3 in culture and contractility measurement was carriedout on that day.

C. Determination of Contractile Amplitude

Measurement of contractile amplitude in cultured ventricular cells wascarried out according to the methods of Xu, D., Kong, H., Liang, B.T.,Circ. Res. 70:56-65 (1992), and Barry, W.H., and Smith, T.W., J.Physiol. (Lond) 325:243-260 (1982). Ventricular myocytes became adheredto coverslips at the bottom of the dish during culturing, and exhibitedspontaneous rhythmic beating by day 3 of culturing. Coverslipscontaining beating cells were placed in a perfusion chamber situated onthe stage of an inverted phase-contrast microscope (Nikon) with an inletand an outlet which allowed infusion and removal of medium containingthe various adenosine analogs. The contractile amplitude of the culturedcell was determined by an optico-video motion detection system with avideo motion analyzer (Colorado Video, Boulder, Colo.) as previouslydescribed. The perfusion medium contained the various adenosine analogsindicated as well as the following components (mmol/L): HEPES 4(pH=7.4), NaCl 137, KCI 3.6, MgCl₂ 0.5, CaCl₂ 0.6, glucose 5.5 and horseserum at 6%. Measurement of contractile amplitude was carried out ononly one cell per coverslip and each culture dish contained 5coverslips. After achieving a steady state of beating in medium withoutadenosine analogs, the medium was switched to that containing theindicated adenosine drugs. Both the basal contraction amplitude and theamplitude measured during adenosine analog exposure were determined. Thestimulatory effect of the various adenosine analogs on the contractilestate was predominantly on the amplitude of contraction (see Xu, D.,Kong, H., and Liang, B.T., supra). The basal rate of contraction was105±16, n=311, ±S.D. There was no significant consistent effect of anyof the analogs on the spontaneous rate of contraction.

It can be seen from FIGS. 1a-1d that the P2_(y) agonist 2-methylthio ATPwas capable of stimulating an increase in contractile amplitude, andthat P2_(x) (α,β-methylene ATP) and P2_(u) (UTP) agonists were notcapable of producing the same effect. These data indicate that a P2_(y)-like receptor is responsible for mediating a positive inotropicresponse.

EXAMPLE 3

Intact myocyte binding with the P2_(y) -selective radioligand ³⁵S!5'-O-2-thiodiphosphate ( ³⁵ S!ADPβS).

Cardiac ventricular myocytes were prepared and incubated withprogressively increasing concentrations of ³⁵ S!ADPβS. The bindingreaction is performed by the addition of a Dulbecco's Modified Eagle'sMedium (which contains 1-glutamine and glucose but lacks phenol red,adenosine nucleotides, and sodium bicarbonate, buffered by HEPES,pH=7.4) containing ³⁵ S!ADPβS (0.5 to 200 nM, for saturation isothermstudy). Nonradioactive ADPβS is used to define the level of nonspecificbinding. Additional purinergic agonists or antagonists are addeddepending on the experimental conditions. Following a 30-minuteincubation at 37° C., cells are washed three times with 3 ml of ice-coldwash buffer (containing 120 mM NaCl, 5.4 mM KCI, 0.8 mM MgSO₄, 1.8 mMCaCl₂, 50 mM HEPES, and 1.0 mM NaH₂ PO₄ adjusted to pH=7.4). One ml of0.5M NaOH is added to the monolayer culture to solubilize the cellprotein and 0.5 ml of Tris buffer, pH=7.4 is added to neutralize NaOHprior to scintillation counting for ³⁵ S.

To determine the B_(max) and K_(d) of the specific ³⁵ S!ADPβS binding, acomputer-aided nonlinear regression analysis (LIGAND PROGRAM) is used.See, Annals of Biochemistry 1980, 107, 220-239. Both a one-site and atwo-site model is applied to fit the data points. The data indicate that³⁵ S!ADPβS labelled both high- and low-affinity sites in the intactmyocyte binding and that over the range of 0.5 to 12 nM, the radioligandlabels the high-affinity sites with a linear Scatchard plot; whereasover the range of 150-150 nM, a low-affinity site (K_(d) =40 nm) islabeled which has a high B_(max) (in the range of 1000 fmole per mg oftotal cellular proteins).

FIG. 2a represents a saturation isotherm of ³⁵ S!ADPβS binding to intactmyocytes and FIG. 2b shows a Scatchard transformation of the data inFIG. 2a. The low affinity and high affinity binding sites for ³⁵ S!ADPβSare readily seen. Three lines of evidence exist which indicate that thehigh-affinity sites mediate the positive inotropic response: (1) theorder of potency of various agonists in causing the positive inotropiceffect is similar to the order of potency of the same agonists incompeting against the high-affinity ³⁵ S!ADPβS sites; (2) thedesensitization of the positive inotropic effect of ATP agonists iscorrelated with the disappearance of the high-affinity ³⁵ S!ADPβS sites;and (3) the EC₅₀ values of the agonists in producing the positiveinotropic effect are similar to the Ki values of the same agonists incompeting with the high-affinity ³⁵ S!ADPβS sites.

EXAMPLE 3

Structure-Activity Relationships

Part I: A series of compounds were tested for the ability to stimulatean increase in myocyte contractility. The ability to produce a positiveinotropic response was compared to the ability to inhibit thehigh-affinity binding sites for ³⁵ S!ADPβS. FIG. 3 shows the EC₅₀ valuesdetermined for each agonist stimulating myocyte contractility comparedto the K_(i) of the same agonists in inhibiting high-affinity ³⁵ S!ADPβSbinding. It can be seen that in general, the five compounds testedexhibit a strong positive inotropic responses which closely correspondto their Ki values.

Part II: A second series of compounds were treated as in part I above.Although the EC₅₀ values for these compounds closely correspond to theirKi values, it can be seen from FIG. 4 that these compounds have amarkedly diminished ability to stimulate myocyte contractility or toinhibit high-affinity ³⁵ S!ADPβS binding.

EXAMPLE 4

Use of Cultured Chick Ventricular Cells and Adult Rat CardiacVentricular Cells as Models for the Characterization of Cardiac P2_(y)Purinergic Receptor

Ventricular myocytes cultured from chick embryos 14 days in ovo andcells isolated from adult rat heart ventricles were used as novel cellmodels to characterize the cardiac P2PR. ATP caused a large increase inthe contractile amplitude (maximal % increase =89.7±9%, n=14±SE), whichwas determined via a video motion detection system. ADP (47.7±10%, n=8),AMP (9.6±4%, n =7) and adenosine (15±4%, n =24) were much lessefficacious. To determine the subtype of P₂ PR involved, the ability ofagonists selective at the P2_(y) (ADPβS and 2-methylthio ATP), P2_(x)(α,β-methylene ATP) and P2_(u) (UTP) receptors to increase contractileamplitude were determined. The maximal percent increase wasADPβS >2-methylthio ATP (76±15%, n=7 and 54±7%, n=17,respectively) >>UTP (22±4%, n=7) or α,β-methylene ATP (-13 ±5%, n=7).Prior exposure of the myocytes to 100 μM of 2-methylthio ATP for 60minutes desensitized the positive inotropic response to ATP, ADPβS andUTP, whereas pretreatment of the myocytes with 100 μM UTP for threehours failed to cause such desensitization. These data validate the useof these cardiac cells as a model for the study of the cardiac receptor,and indicate that a P2_(y) -like purinergic receptor mediates thepositive inotropic response of ATP.

EXAMPLE 5

Synthesis of Compounds

A. Synthesis of compounds with 3'-modified ribose modifications

3'-amino-3'-deoxyadenosine 5'-triphosphate and potassium carbonate aredissolved in water. 3-(4- Hydroxyphenyl) ethylene N-hydroxysuccinimideester, 3-(4-hydroxy) phenylic N-hydroxysuccinimide ester, or3-(4-hydroxyphenyl) pentylene N-hydroxysuccinimide ester indimethylsulfoxide is added and the mixture is stirred at roomtemperature for 24 hours. The product is purified by repeated injectionon HPLC using a Synchropak RP-100 column applying a linear gradient ofacetonitrile 5-22% TEAA. The fractions are collected and lyophilized todryness. The product is obtained as a triethylammonium salt. Derivativesof 3'-amino-3' deoxy-ATP are prepared by acylation of the amino group orits reductive alkylation with alkyl/aryl aldehyde and cyanoborohydride,as indicated in FIG. 5. See Burnstock, G., et al., Drug DevelopmentResearch 31:206-219, 1994.

B. Synthesis of 2-substituted ATP derivatives (FIGS. 6a and 6b)

The starting material, adenosine N-oxide, is prepared as according tothe procedure of Kikugawa, K., et al., A. Chem. Pharm. Bull. 25:1959,1979. Adenosine N-oxide is added to a solution of 5M NaOH, refluxed onan oil bath for 15 minutes and then rapidly chilled in ice-water and indry-ice acetone. The pH is adjusted to 9 and the solution is evaporatedon a rotary evaporator. The residue is taken up in methanol, andprecipitated NaCl is removed by pressure filtration through frittedglass, which is washed with methanol and then evaporated. The product isthen dissolved in water. The aqueous solution is mixed with methanol andcarbon disulfide and heated in a pressure and heat-resistant vessel. Theresulting 2-thioadenosine is then reacted with NaOH and then with tenequivalents of alkyl-aryl halide in the presence of ethanol. Thereactions are evaluated by TLC (silica, CHCl₃ /methanol 85:15 or CHCl₃/methanol/acetic acid 85:10:5). Once complete the product iscrystallized by evaporation. At this point, the various 2-alkyl or arylthioadenosine derivatives will be phosphorylated to the correspondingnucleoside mono-, di-, or tri-phosphates according to the method ofKovacs, T., et al., Tetrahedron Letters, 29(36): 4525-4528, 1988. Thevarious nucleoside phosphates are isolated by HPLC using a gradient from0 to 15% acetonitrile in 50 mM ammonium formate. The2-alkyl/aryl-thioadenosine tri-phosphates will have a retention timeranging from 7 to 14 minutes.

Several N⁶ -substituted adenosine and 2-substituted derivatives whichinclude a phenyl group are commercially available can be phosphorylatedto the corresponding nucleoside mono-, di-, and triphosphates by theprocedure of Kovacs, T., et al., supra.

C. Synthesis of 2,6-disubstituted ATP derivatives

The third class of compounds include substitution at both the N⁶position and the 2-position. The starting compounds are the N⁶-substituted derivatives, for example N⁶ -benzyladenosine, N⁶-phenyladenosine or 2-phenylaminoadenosine, or N⁶ -methyladenosine whichis also commercially available. These are first N-oxidized usingm-chloroperbenzoic acid in acetic acid, and the N⁶ -substitutedadenosine N-oxide is then substituted at the 2-position by a thio group,followed by alkylation with alkyl halide in NaOH to produce a2-alkyl/aryl-thioadenosine derivative with substitution at the N⁶position. Such adenosine derivatives are phosphorylated to yield thecorresponding nucleoside mono-, di-, and tri-phosphates according to themethod of Kovacs, T., et al., supra. Groups suitable for 2-alkyl oraryl-thioadenosine substitution include all those outlined in FIGS. 6aand 6b.

Those skilled in the art will appreciate that numerous changes andmodifications may be made to the preferred embodiments of the inventionand that such changes and modifications may be made without departingfrom the spirit of the invention. It is therefore intended that theappended claims cover all such equivalent variations as fall within thetrue spirit and scope of the invention.

What is claimed is:
 1. A compound having structure: ##STR3## or a pharmaceutically acceptable salt thereof, wherein: R₁ and R₂, independently, are halogen or --R₆ --(R₇)_(p) --R₈ ;R₃ is halogen or --R₆ --(R₇)_(p) --R₈ ; R₄ is OH or SH; R₅ is OH or acetamido; R₆ is NH or S; R₇ is alkylene having from 1 to 10 carbon atoms; R₈ is H, NH₂, CN, cycloalkyl having 3 to about 10 carbon atoms, or aryl having 3 to about 20 carbon atoms; X and Y are independently N or CH; n is 0 or 1; and p is 0 or
 1. 2. The compound of claim 1 wherein R₈ is --C₆ H₁₁, --C₅ H₉, --C₆ H₅,--C₆ H₄ --NO₂, or --CH C₆ H₅ (CH₃)! C₆ H₅ (OCH₃)₂ !.
 3. The compound of claim 1 wherein R₁, R₂, or R₃ is --NH₂, --NH--CH₃, --NH--C₂ H₅, --Cl, --S--CH₃, --NH--CH₂ --NH₂, --NH--(CH₂)₂ --NH₂, --NH--(CH₂)₃ --NH₂, --NH--(CH₂)₄ --NH₂, --NH--(CH₂)₅ --NH₂, --NH--(CH₂)₆ --NH₂, --S--(CH₂)₆ --CN, --S--CH₂ --C₆ H₄ --NO₂, --S--(CH₂)₂ --C₆ H₄ --NO₂, --S--CH₂ --CN, --S--(CH₂)₂ CH, --S--(CH₂)₃ CN, --S--(CH₂)₄ CN, --S--(CH₂)₅ CN, NH--C₆ H₅, NH--CH₂ --C₆ H₅, NH--(CH₂)₂ --C₆ H₅, NH--(CH₂ (CH₂)₅, --NH--(CH₂)₆ --C₆ H₅, --NH--C₆ H₁₁, --NH--C₅ H₉, NH--CH₂ --CH C₆ H₄ (CH₃)! C₆ H₃ (OCH₃)₂ !.
 4. The compound of claim 1 wherein R₄ and R₅ are OH; X and Y are N; and n is
 1. 5. The compound of claim 4 wherein R₁ and R₂ are NH₂.
 6. The compound of claim 4 wherein R₁ and R₂ are S.
 7. The compound of claim 4 wherein R₁ and R₂ are Cl.
 8. The compound of claim 4 wherein R₁ is Cl; R₂ is S.
 9. The compound of claim 4 wherein R₁ is S and R₂ is Cl.
 10. The compound of claim 1 wherein R₄ is S; R₅ is OH; X and Y are N; and n is
 0. 11. The compound of claim 10 wherein R₁ and R₂ are NH₂.
 12. The compound of claim 10 wherein R₁ and R₂ are S.
 13. The compound of claim 10 wherein R₁ and R₂ are Cl.
 14. The compound of claim 10 wherein R₁ is Cl and R₂ is S.
 15. The compound of claim 10 wherein R₁ is S; R₂ is Cl.
 16. The compound of claim 12 wherein R₄ is OH; R₅ is acetamido; X and Y are N; and n is
 1. 17. The compound of claim 1 wherein R₄ is OH; R₅ is OH; X is CH; Y is N; and n is
 1. 18. The compound of claim 1 wherein R₄ is OH; R₅ is OH; X is CH; Y is N; and n is
 0. 19. The compound of claim 1 wherein R₄ is OH; R₅ is acetamido; X is C; Y is N; and n is
 1. 20. The compound of claim 1 wherein R₄ is OH; R₅ is acetamido; X and Y are C; and n is
 1. 21. The compound of claim 1 wherein R₄ is S; R₅ is acetamido; X is C; Y is N; and n is
 0. 22. The compound of claim 1 wherein R₄ is S; R₅ is acetamido; X and Y are C; and n is
 0. 23. A composition comprising the compound of claim 1 and a pharmaceutically acceptable carrier, adjuvant, or vehicle. 